This invention relates to a method for the determination of triglycerides. More particularly, this invention relates to a method for the determination of triglycerides in biological fluids with an oxygen-sensing electrode which measures dissolved oxygen in solution.
The triglycerides, or glycerol esters of fatty acids, are important water-insoluble substances found in plants and animals and comprise a major portion of the lipids in the human body. Although dietary fats and oils are the chief sources of lipids in the body, lipids are also synthesized in the body from nonlipid precursors. The importance of these fats in the diet can be attributed, in part, to the fact that they are the most concentrated of all food materials, furnishing about 9 calories of energy per gram as sources of heat and energy. Fats also promote the more efficient utilization of calories supplied by other foods, provide increased palatability and small amount of flavoring to other foods, and are vehicles for the fat-soluble vitamins A, D, E and K.
Digested fats are normally absorbed into the lymph through the duodenum and jejunum. About 100 grams of exogenous glycerides are absorbed by the intestinal mucosa daily in an average healthy person and transported as chylomicrons into the blood stream. Together with cholesterol and phospholipids, the triglycerides (neutral fats) constitute the principal lipids occurring in the blood.
Cholesterol, phospholipids and triglycerides are transported in the blood as macromolecules in combination with plasma globulins as lipoproteins, which also include the unesterified fatty acids bound to plasma albumin. An increase of any one or a combination of these lipids in the blood serum may undesirably increase the total lipids (hyperlipemia) whereas a decrease of the same may undesirably decrease the total lipids (hypolipemia).
In recent years, considerable attention has been given to the relationship between elevations of serum lipids and an increased risk of coronary artery disease and atherosclerosis. Since dietary and medical therapeutic means are now available to reduce serum lipid levels, methods for the determination of serum triglycerides are of significant clinical interest.
Historically, serum triglycerides have been determined generally by various macro- and micromethods such as described by Bloor, "Biochemistry of the Fatty Acids," ACS Monograph No. 93, Reinhold Publishing Company, New York, at pages 37-49 (1943). These procedures make use of the known phenomenon that triglycerides are readily soluble in the so-called fat solvents, for example, ether, petroleum ether, chloroform, carbon disulfide and carbon tetrachloride, but only sparingly soluble in alcohol. According to these procedures, the triglycerides are extracted from blood with the fat solvent, typically ether. Due to the presence of a high percentage of protein, it is generally necessary to follow the original ether extraction by extraction with alcohol followed by a second ether extraction. After extraction, the solvent is stripped by evaporation at low temperature, which leaves behind the remaining sample of triglycerides for examination and quantitation.
More recently, blood glycerol levels have been used as an index of lipolytic events in the body and various methods have been developed for determining these glycerol levels chromatographically, colorimetrically, enzymatically and fluorimetrically. In these procedures, the triglycerides are first saponified such as by alcoholic KOH and then the liberated glycerol is determined. In one typical procedure developed by Wieland, Biochem. Z.329, 313 (1957), glycerol is enzymically converted by glycerokinase (GK) to .alpha.-glycerophosphate (.alpha.-GP) which, with .alpha.-glycerophosphate dehydrogenase (.alpha.-GPDH) and nicotinamide adenine dinucleotide (NAD), gives reduced nicotinamide adenine dinucleotide (NADH). The latter compound is then measured spectrophotometrically at 340 nm. A modification of this procedure described by Eggstein, Klin. Wscht. 44, 262 (1966), employs adenosine-5'-triphosphate (ATP), NADH, phosphoenolpyruvate (PEP), GK, pyruvate kinase (PK) and lactate dehydrogenase (LDH) in the system in which the decrease in NADH content is similarly measured spectrophotometrically. Yet another procedure reacts glycerol with NAD and glycerol dehydrogenase followed by spectrophotometric determination of the resulting NADH. These and similar such procedures are further described in Henry, "Clinical Chemistry, Principles and Technics", Harper and Row, Publishers 1964; Richterich, "Clinical Chemistry", Academic Press, Publishers 1969; Tietz, "Fundamentals of Clinical Chemistry," W.B. Saunders Company, Publishers 1970, and references cited therein.